Dengue is the most important arthropod-borne viral disease in the world. No effective vaccines are available and mosquito control remains the only prevention option. Transmission of dengue virus to a human host is totally dependent upon availability of a competent mosquito vector, usually Aedes aegypti. It is well known that microbes in the insect gut play a very important role in the biology of insects, and can influence growth of some insect pathogens. Considerable effort is being directed toward applying transgenesis technology to arthropod-borne disease control, with an emphasis on population replacement. A major focus is to use these techniques to generate mosquitoes that carry genes that disrupt their ability to transmit pathogens. Another possibility is paratransgenesis, wherein bacteria capable of colonizing the mosquito midgut would be used to carry and express anti-pathogen effector genes and thereby prevent disease transmission to humans by the mosquito. The present knowledge on normal midgut bacterial flora in Ae. aegypti and the potential effects on fitness is limited. The long-term objectives of this project are to develop an understanding of the interrelationships between Ae. aegypti populations and their natural midgut bacterial flora. This research will be done primarily in India at Pune University in collaboration with Dr. Yogesh Shouche, as an extention of NIH Grant No. RO1-AI059342. The specific aim of this project is to: Investigate the role of midgut bacteria on fitness in Ae. aegypti. This includes efforts to: 1) compare midgut bacterial flora of adult Ae. aegypti in laboratory strains and field isolates, 2) examine the impact of midgut bacteria on Ae. aegypti life history traits, and 3) determine the effect of midgut bacteria on dengue virus susceptibility in Ae. aegypti. Bacterial identity will be determined by amplification and sequencing of the 12S and 16S rRNA genes from both culturable bacteria and total midgut contents, which will include unculturable species. The impact of midgut bacteria on fitness will be examined among antibiotic treated adults for several life history traits including development time, adult body size, bloodmeal size, fecundity, fertility, and adult longevity. Dengue virus susceptibility of females from dengue refractory and susceptible Ae. aegypti colonies will be compared among normal and antibiotic treated individuals. PUBLIC HEALTH RELEVANCE: Dengue is a threat to >2.5 billion people, with estimates of 50 million cases of dengue fever and ~24,000 deaths due to the more severe hemorrhagic form each year. No vaccines are available and disease prevention is dependent on controlling the mosquito vector, primarily Aedes aegypti. The proposed research will provide valuable new information regarding the influence of midgut bacteria on the biology of the mosquito, including the ability to support and transmit the dengue virus to humans.